The goal of the proposed studies is to understand how DNA polymerases discriminate between right and wrong dNTPs. In order to better understand how polymerases discriminate between correct and incorrect dNTPs, the interaction of human DNA polymerase alpha with dNTPs will be examined. The specific aims of this proposal are: (1) Develop a complete understanding of how pol alpha discriminates between right and wrong dNTPs. The specific functional groups on a dNTP with which pol alpha interacts will be defined. (2) evelop novel base-pairs that pol alpha replicates with high fidelity and efficiency. To accomplish these goals, a series of dNTP analogs will be synthesized and their interactions with pol alpha determined using steady-state kinetic assays. DNA replicaiton inhibitors comprise a major class of anti-cancer and anti-viral chemotherapeutics, with approximately 2 dozen clinically useful compounds and many more in various stages of development. Thus, a better understanding of the enzymes involved in DNA replication could lead to the development of new chemotherapeutics. A key question regarding DNA and RNA polymerases is how they discriminate between correct and incorrect (d)NTPs. Since DNA polymerase-mediated mutagenesis can lead to cellular transformation, understanding how DNA polymerases discriminate between correct and incorrect dNTPs may lead to a more fundamental understanding of carcinogenesis. This research will be done primarily in the Czech Republic in collaboration with Dr. Michal Hocek as an extension of NIH grant # R01AI059764.